Clot formation and dissolution are essential to human survival. Abnormal clot formation and occlusion of blood vessels of the heart, lung, brain, and peripheral circulation can result in death, incapacitation, and limb amputation. Rapid treatment with thrombolytic agents to dissolve clots and restore blood flow is a means to minimize morbidity and mortality in these patients. In the USA, more than 1 million patients are affected annually by blood clots in the heart, brain, lung or peripheral vessels, with more than 600,000 deaths occurring from myocardial infarction and ischemic stroke. This costs the US economy in excess of $40 billion per year. This proposal is to assess the pre-clinical efficacy of a direct thrombolytic agent delivered locally in a rabbit model of abdominal aorta thrombosis. Active plasmin is generated from a stable depot form of plasmin, AZ-plasmin, by exposure to energy (light, sound waves). Plasmin is a proteolytic enzyme that digests fibrin and degrades clots, releasing trapped blood cells and re-establishing circulation. Local delivery of plasmin and its rapid inhibition by alpha2 antiplasmin distal to the clot site will limit bleeding side effects. Current thrombolytic therapies infuse protein activators (streptokinase, tissue plasminogen activator) to convert plasminogen to plasmin; plasminogen concentrations become limiting in areas of diminished blood flow. These therapies do not achieve their full potential due to significant side effects, including increased risk of hemorrhage and stroke. The proposed specific aims are (1) to optimize the preparation of AZ-plasmin from human plasmin and a proprietary inhibitor, AZ-nu, and determine the properties of AZ-plasmin and light-reactivated plasmin, using a synthetic substrate; (2) to characterize delivery and activation parameters for AZ-plasmin in plasma clots and blood clots in vitro, using catheters and fiber optic light or shock wave energy sources to activate AZ-plasmin to plasmin; and (3) to determine safety and efficacy of AZ-plasmin activated to plasmin for restoring blood flow in vivo in rabbits with abdominal aorta thrombosis, a model for peripheral arterial occlusion. These experiments will establish the utility of AZ-plasmin as a stable, effective source of the direct thrombolytic agent plasmin for treatment of occlusive thrombi, with the potential for a better safety profile and fewer side effects than current therapies.